Histochemical evidence of three types of esterases in the adrenal medulla of the rat

Summary Esterases of the adrenal medulla have been studied histochemically using alpha-naphthyl acetate and butyrate as substrates, Blue RR Salt as a coupler and eserine and E, 600 as inhibitors. Three types of esterase activity were thus demonstrated: (1) cholinesterase activity in the nerve fibres, ganglion cells and secretory medullary cells; (2) eserine resistant but E. 600 sensitive esterase activity in the ganglion cells and secretory cells; (3) E. 600 resistant activity in strongly positive, unidentified cells scattered in the medulla. The histochemical picture was essentially similar in sections of formalin-fixed tissue and in fresh sections subjected to the voltage gradient employed for electrophoretic separation of esterases. It is concluded that esterases histochemically demonstrable in sections are desmo-enzymes and at least to a major part different from the lyo-enzymes which can be separated by starch gel electrophoresis.With 6 Figures in the Text     

Histochemical patterns of lipolytic enzymes of the hepatopancreas of Scylla serrata and their possible relation to eyestalk factor(s)

Of the lipolytic enzymes studied histochemically, lipase (Tween 80 specific enzyme) showed highest activity while non-specific esterases (Tween 60, α-naphthyl acetate, β-naphthyl acetate and 5-bromo-indoxyl acetate esterases) and Tween 20 and Tween 40 esterases exhibited medium and lowest activities respectively. The pattern of distribution of these enzymes was found to be variable among the various elements of the hepatopancreas. Lipase. Tween 20 and Tween 40 esterases were localized at the apical parts of the R-cell, F-cell cytoplasm, the B-cell vacuolar contents and the lumina of the hepatopancreatic acini. Non-specific esterases were primarily present in the cytoplasm of the R-cells, brush border of the tubules and connective tissue. A striking reduction in the lipase activity of the R- and B-cells was apparent 4 hours after the bilateral extirpation of eyestalks. Restoration of activity was observed 48 hours after the operation. On the other hand, a rise in the level of non-specific esterases was conspicuous 4 hours after the eyestalk amputation. This increased enzyme activity was restored to normal 24 hours after the operation. Administration of eyestalk extract into normal and de-stalked animals caused an increase in the lipase activity of the R- and B-cells. Surprisingly, the activity of non-specific esterases also enhanced considerably in the R-cell cytoplasm and connective tissue. The enhanced activity of lipase and non-specific was noted 4 hours after the treatment. From the present findings it appears that the eyestalk hormone(s) are directly involved in regulating the activity of the lipolytic enzymes. The physiological role of the F- and B-cells seems to be secretion and extrusion of lipase respectively. The R-cells and connective tissue appear to be associated with storage and mobilization of lipids.     

Distribution tissulaire des estérases chez l'etourneau sansonnet (Sturnus Vulgaris)

Tissue distribution of esterase was examined by electrophoresis in the blood, liver and heart tissues of the starling Sternus vulgaris. Esterases have been found in all tissues examined but the profile varies from tissue to tissue, depending on the absence or presence of particular isozymes, as well as the relative activity of these that are present. Overall, liver has the most esterase activity; red cells and plasma, liver tissue and heart tissue have distinctly different esterases.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Catalytic histochemistry of acid and neutral hydrolases in plant seedlings

Summary In contrast to human and animal tissues, little information is available on the activity, distribution and functional role of acid and neutral hydrolases in plant cells and tissues. Because it is known that these enzymes are relatively active during germination, they were analysed histochemically during this process using light microscope azo, azoindoxyl, indigogenic and tetrazolium methods. Proteases, glucosidases and glucuronidases could not be detected. Non-specific acid phosphatases were species-independent and showed considerable activities in aleuron and nutritional cells, in other cell types of cotyledon or endosperm tissue and in different types of embryonic cells. Acid glycosidases and non-specific esterases, in contrast displayed a species-dependent activity and differences in localization. Of the glycosidases, -d-galactosidase was the most active. Non-specific esterases, acid phosphatase and glucosaminidase were also present in the extracellular matrix. During germination, acid hydrolase activity either decreased or increased, depending on the seedling species and enzyme.     

Nature of human spleen red pulp cells with special reference to sinus lining cells

Summary A histological and cytological as well as enzyme histo- and cytochemical analysis (alpha-naphthyl acetate esterases, naphthol AS acetate esterases, naphthol AS-D chloroacetate esterases, acid and alkaline phosphatases) of human spleen cells in sections and imprints was carried out with special reference to sinus lining cells. These cells show strong naphthol AS esterase activity and no or only little alpha-naphthyl acetate esterase and acid phosphatase activity. Thus they can be distinguished from reticular cells in pulp cords and from other macrophages in cords and sinuses. From the morphological and enzyme histochemical aspect it can be deduced that the sinus lining cells are a distinct cell type of the human spleen. The comparison of these enzyme cytochemical findings with the results of biochemical and electron microscopical investigations suggests that reticular cells of pulp cords and littoral cells of sinuses also have different functions: reticular cells seem to have a high phagocytotic activity while littoral cells seem to be only facultatively phagocytic.This work was supported by the Deutsche Forschungsgemeinschaft.     

The non-specific esterases of mouse lung

The non-specific esterases of the lung of the house mouse, M. musculus, were examined by polyacrylamide electrophoresis and by isoelectric focusing. At least 13 different esterases were distinguished and identified, mainly by their catalytic properties, susceptibility to inhibition, developmental patterns and phenotypic variation amongst different strains. A list of diagnostic features of the 13 esterases was presented. None of the esterases was lung-specific. However, the pattern of esterases found in the adult lung was characteristic of that organ. It was pointed out that this pattern is associated with the high degree of tissue differentiation in the adult lung. At least 8 esterases were found which belong to the isozyme system of carboxylesterase EC 3.1.1.1, under the control of genes located on chromosome 8. These esterases accounted for about 90% of the esterase activity in the lung.     

A light and electron microscopic investigation of the digestive system of the Ophiuroid ophiuroiderma panamensis (Brittle Star)

The Brittle Star digestive system is composed of buccal, pharyngeal, esophageal and stomach cavities. The buccal and pharyngeal cavities are lined by columnar cells covered by a cuticle, and are apparently concerned with mucous production. Coelomocytes and tall columnar cells are described in the esophagus and stomach epithelia. The columnar cells are adapted for nutrient absorption, enzyme synthesis, and lipid storage. Nerves are found beneath the epithelia within a connective tissue layer. Smooth muscle and coelomic layers lie external to the connective tissue layer. The coelomic layer lines a perivisceral space and has diverse modifications of its perivisceral surface; a pedicle-cuticle modification perhaps having general significance in echinoderms.     

Effects of rosiglitazone and high fat diet on lipase/esterase expression in adipose tissue

A number of intracellular lipase/esterase have been reported in adipose tissue either by functional assays of activity or through proteomic analysis. In the current work, we have studied the relative expression level of 12 members of the lipase/esterase family that are found in white adipose tissue. We found that the relative mRNA levels of ATGL and HSL are the most abundant, being 2-3 fold greater than TGH or ADPN; whereas other intracellular neutral lipase/esterases were expressed at substantially lower levels. High fat feeding did not alter the mRNA expression levels of most lipase/esterases, but did reduce CGI-58 and WBSCR21. Likewise, rosiglitazone treatment did not alter the mRNA expression levels of most lipase/esterases, but did increase ATGL, TGH, CGI-58 and WBSCR21, while reducing ADPN. WAT from HSL-/- mice showed no compensatory increase in any lipase/esterases, rather mRNA levels of most lipase/esterases were reduced. In contrast, BAT from HSL-/- mice showed an increase in ATGL expression, as well as a decrease in ES-1, APEH and WBSCR21. Analysis of the immunoreactive protein levels of some of the lipases confirmed the results seen with mRNA. In conclusion, these data highlight the complexity of the regulation of the expression of intracellular neutral lipase/esterases involved in lipolysis.     

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio)

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.     

Esterases as pesticide biomarkers in crayfish (Procambarus clarkii, Crustacea): tissue distribution, sensitivity to model compounds and recovery from inactivation

The specific activities of acetyl- and butyrylcholinesterase and carboxylesterase were assayed in the digestive gland and in nervous and muscle tissues of the crayfish Procambarus clarkii. Since acetylcholinesterase prevails in nervous tissue and carboxylesterase in digestive gland, they are proposed as biomarkers. Muscle had negligible activities of all esterases, and all tissues had a low butyrylcholinesterase activity. Esterases were mostly cytosolic in digestive gland and muscle, but membrane-bound in nervous tissue; use of Triton X-100 is not recommended due to its widely diverging effects in esterase assays. Phenylmethylsulphonylfluoride inhibited acetyl- and butyrylcholinesterase in extracts from all tissues, and in digestive gland only carboxylesterase. In digestive gland, tetra[monoisopropyl]-pyrophosphorotetramide inhibited all esterases with different sensitivities, while in muscle and nervous tissue it only partially inhibited all esterases. Carbamates inhibited 100-fold more strongly than organophosphates acetyl- and butyrylcholinesterase activities. Carboxylesterase was inhibited by carbaryl and chlorpyrifos, but not by eserine and malathion. In vitro conditions to evaluate recovery from inactivation of esterases by model pesticides were established for acetylcholinesterase and carboxylesterase. The new reactivation protocol could be useful as a biomarker of pesticide exposure to differentiate between dilution-reversible inhibitions, indicating carbamate exposure, from dilution-irreversible effect, attributed to organophosphate exposure.     

ENZYMES OF THE LUNG : I. Detection of Esterase with a New Cytochemical Method

The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mµ permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.     

Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.     

Electron microscopic study on the gill bars of amphioxus (Brachiostoma californiense) with special reference to Neurociliary control

Summary Both primary and secondary (tongue) bars of the pharyngeal gill basket are covered by epithelial cells that are continuous with the cells that line the atrium. Anterior and posterior faces of the gill bars are covered with lateral ciliated cells, which possess a single cilium, ringed by microvilli, and an elaborate basal mitochondria-rootlet apparatus. Pharyngeal faces of the gill bars are covered with ciliated pharyngeal cells, atrial faces by mucus secreting atrial cells. The surface epithelium rests on a stromal septum, a flattened tube of basal lamina which dilates to form the visceral blood vessel (along the pharyngeal face) and skeletal blood vessel (along the atrial face). This basal lamina surrounds paired skeletal rods which run through the longitudinal axis of the gill bars near the atrial face. Between the skeletal rods and atrial cells of primary gill bars is a coelomic channel lined by epithelioid coelomic cells. Neuronal processes, some with neurosecretory granules, are located among the bases of the atrial cells. Some axons may contact lateral ciliated cells where the latter meet atrial cells, but synaptoid endings have not been found here or elsewhere in the gill bars. Nervous tissue has not been identified among lateral ciliated cells even though ciliary activity of these cells is supposedly regulated by atrial nervous tissue.Supported by a Cottrell College Science Program Grant from Research Corporation. We thank Nancy Kelly and Gerhard Ott for excellent technical assistance and are grateful for the facilities provided by the Department of Zoology and Seaver Science Center, Pomona College.     

Evidence of a true pharyngeal tonsil in birds: a novel lymphoid organ in Dromaius novaehollandiae and Struthio camelus (Palaeognathae)

ABSTRACT: BACKGROUND: Tonsils are secondary lymphoid organs located in the naso- and oropharynx of most mammalian species. Most tonsils are characterised by crypts surrounded by dense lymphoid tissue. However, tonsils without crypts have also been recognised. Gut-associated lymphoid tissue (GALT), although not well-organised and lacking tonsillar crypts, is abundant in the avian oropharynx and has been referred to as the "pharyngeal tonsil". In this context the pharyngeal folds present in the oropharynx of ratites have erroneously been named the pharyngeal tonsils. This study distinguishes between the different types and arrangements of lymphoid tissue in the pharyngeal region of D. novaehollandiae and S. camelus and demonstrates that both species possess a true pharyngeal tonsil which fits the classical definition of tonsils in mammals. RESULTS: The pharyngeal tonsil (Tonsilla pharyngea) of D. novaehollandiae was located on the dorsal free surface of the pharyngeal folds and covered by a small caudo-lateral extension of the folds whereas in S. camelus the tonsil was similarly located on the dorsal surface of the pharyngeal folds but was positioned retropharyngeally and encapsulated by loose connective tissue. The pharyngeal tonsil in both species was composed of lymph nodules, inter-nodular lymphoid tissue, mucus glands, crypts and intervening connective tissue septa. In S. camelus a shallow tonsillar sinus was present. Aggregated lymph nodules and inter-nodular lymphoid tissue was associated with the mucus glands on the ventral surface of the pharyngeal folds in both species and represented the Lymphonoduli pharyngeales. Similar lymphoid tissue, but more densely packed and situated directly below the epithelium, was present on the dorsal, free surface of the pharyngeal folds and represented a small, non-follicular tonsil. CONCLUSIONS: The follicular pharyngeal tonsils in D. novaehollandiae and S. camelus are distinct from the pharyngeal folds in these species and perfectly fit the classical mammalian definition of pharyngeal tonsils. The presence of a true pharyngeal tonsil differentiates these two ratite species from other known avian species where similar structures have not been described. The pharyngeal tonsils in these ratites may pose a suitable and easily accessible site for immune response surveillance as indicated by swelling and inflammation of the tonsillar tissue and pharyngeal folds. This would be facilitated by the fact that the heads of these commercially slaughtered ratites are discarded, thus sampling at these sites would not result in financial losses.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies     

The distribution of carboxylic esterases in the area postrema of the guinea pig

Summary Detailed studies were made on the distribution of carboxylic esterases in the area postrema (AP) of the guinea pig. In this species the nerve cells of the AP show intense acetylcholinesterase activity which greatly enhances a morphological study of these elements. The two main types of nerve cells arebipolar cells andmultipolar cells. The bipolar cells often send their peripheral processes to the wall of a capillary, while the other processes go towards the nervous tissues outside of the organ. A similar behavior of the processes could be observed in the case of the multipolar cells. A special multipolar cell having bulb-like endings in the neuropilema of the organ has been discussed. On the basis of these findings it is conjectured that these neural elements, as well as the AChE positive nerve fibers located along the walls of the capillaries, function as parts of a chemoreceptor system, the existence of which has been postulated by former authors. The non-specific esterase activity of the organ is rather diffuse. B esterases have been described in the glial cells and in several nerve cells and in the pericytes and endothelial cells of the perivascular space. The localization of A and C esterases appeared diffuse, but only the AP showed intense C esterase activity in the caudal medullary area.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

Demonstration of hydroxysteroid dehydrogenases and testosterone in the Sertoli-Leydig cell tumor (androblastoma) tissue of the human ovary: an enzyme histochemical and immunohistochemical study

Gynecological, endocrinological and histological tests on a 19-year-old female patient led to the diagnosis of Sertoli-Leydig cell tumor (arrhenoblastoma) of intermediate differentiation. For enzyme histochemical purposes the tumor tissue, removed from the right ovary by laparatomy, was frozen in liquid nitrogen. The following enzymes were demonstrated: nonspecific esterases, 3 beta-hydroxysteroid dehydrogenase (HSDH), 17 beta-HSDH, 11 beta-HSDH, and NADH tetrazolium reductase. Cryostat sections, prefixed with formaldehyde vapors, were used to localize testosterone production immunohistochemically with the PAP method. A large number of pseudotubules with Sertoli cells were observed; the Leydig cells in the interstitial space were often arranged in the form of islands. Strong nonspecific esterase activity weak 3 beta-HSDH activity, moderate 17 beta-HSDH activity, and strong 11 beta-HSDH activity were observed largely in the Leydig cells. Testosterone synthesis, demonstrated immunohistochemically, took place predominantly in the Leydig cells, but also to a small extent in the Sertoli cells.     

The fine structure of the planarianPolycelis tenuis ijima

Summary The fine structure of the pharynx is presented and demonstrates that the pharyngeal epithelial system is a continuous one. The epithelial lining of the pharyngeal cavity with its characteristic fibrous secretory bodies merges with the outer pharyngeal epithelium at the point of anchorage of the pharynx. A few of these cells are insunk, the nuclei occurring beneath the underlying muscular layers. The nature of the outer epithelium changes towards the free end of the pharynx; the cells become ciliated and in contents come to resemble the inner epithelium which it joins at the tip.The gut cells merge at a transitional zone with the inner pharyngeal epithelium and at this point both bear microvilli and contain rod-shaped apical bodies. Some of these cells are also insunk. Towards the mouth the epithelium shows a greater degree of insinking and exhibits microapocrine secretion. Both inner and outer epithelia bear sense receptors which are concentrated at the lip.At the point of pharyngeal insertion, the sub-epithelial tissue resembles planarian parenchyma, but is rich in gland cells. These glands open on to the outer epithelium especially towards the free end of the pharynx.This research was supported by the Scientific Research Council. Grant No. B/RG/086.